Glutamate is the major excitatory neurotransmitter in the brain and the retina. Because of their importance in vision processing, the correct function and localization of glutamate receptors (GluRs) is essential for vision. In addition it has been shown in the brain, that an inappropriate over-stimulation of GluRs leads to neuronal cell death, in a process known as excitatoxicity. Even though this phenomenon has been shown to occur in the retina, its importance has been understimulated. The diversity of GluRs is extensive and subtype specific agonists and antagonists of these receptors have been shown to reduce or enhance excitotoxic cell damage. The goal of the laboratory is to better understand the relationship between the functional and morphological complexity of retinal cell classes and the diversity of GluRs. Specifically, three projects will be pursued: 1) Metabotropic glutamate receptors in retina. At least six metabotropic glutamate receptors, i.e. receptors that activate second messenger pathways upon glutamate stimulation, are expressed in the retina. Using subtype- specific antibodies, we will determine the localization of these receptors in the retina. In situ hybridization and immunohistochemical studies will be done to analyze the pattern of expression of these receptors during development and retinal degeneration, both of which could be regulated by metabotropic receptors. Finally, the possible modulation of voltage- and ligand-gated channels by metabotropic receptors will be analyzed in dissociated retinal neurons in culture. 2) Signal transduction pathway in ON bipolar cells. Since photoreceptor cells hyperpolarize in response to light stimuli, whereas ON bipolar cells depolarize, the response of ON bipolar cells is sign-inverting. This response is probably mediated by a metabotropic glutamate receptor, mGluR6. However, little is known about the second messenger pathway coupled to this receptor. Using a cell sorting procedure, we have prepared RNA from enriched populations of bipolar cells. A cDNA clone encoding a novel putative cGMP-gated channel w as identified from this material and it will be functionally characterized. The same procedure will be used to clone additional components of this pathway, such as G protein subunits. 3) Kainate-type glutamate receptors in retina. Of the three ionotropic glutamate receptor classes, NMDA, AMPA and kainate receptors, the functions of the last are the most poorly understood. We have shown that the expression of several kainate receptor subunit genes is affected by retinal degeneration in the rd mouse. We will raise subunit-specific antibodies against these receptor subunits and analyze these changes at the receptor protein level. By in situ hybridization we have also found that GluR7 has a distinctive pattern of expression during development, since it is transiently expressed in photoreceptors. Making use of very recently constructed transgenic mice,in which the GlluR7 gene is inactivated, we will analyze the role of this receptor subunit in retinal morphology.